Cortical microcircuits, comprising specialized neuron subpopulations and their selective synaptic connections, form functionally segregated output channels to other cortical and subcortical targets. The activity of cortical microcircuits is regulated by a number of modulatory neurotransmitters, such as serotonin (5-HT), that optimize circuit performance for specific cognitive tasks, and which are implicated in a wide spectrum of mental health disorders. For instance, disrupted 5-HT signaling contributes to schizophrenia, depression, and anxiety, while serotonergic drugs are widely used to treat these disorders. However, despite their functional and clinical importance, little is known about how modulatory transmitters selectively regulate the activity of cortical neuron subpopulations subserving specific functional roles within cortical microcircuits. Our long-term goal is to characterize the functional impact of modulatory neurotransmitters on cortical microcircuit function. The research proposed here represents an important first step toward this goal by identifying key cellular and synaptic components of cortical microcircuits differentially regulated by 5-HT. Serotonergic signaling in excitatory cortical neurons relies primarily on two G-protein coupled receptors, 5-HT1A (1A) and 5-HT2A (2A), that have opposing influences on neuron excitability. Based on recent results showing commissural/callosal (COM) projection neurons are selectively excited by 5-HT, our central hypothesis is that endogenous 5-HT acts to selectively enhance the activity of neurons participating in specific executive functions, while suppressing the bulk of cortical output to subcortical structures. Our first aim is to identify the cellular components of cortical circuits that are functionally excited or inhibited by endogenous 5-HT. This will be accomplished in a mouse model in which channelrhodopsin-2 is selectively expressed in 5-HT neurons, and using fluorescent retrograde tracers delivered in vivo. Our second aim is to characterize synaptic connectivity among populations of 5-HT-excited neurons to determine whether they form networks capable of the sustained activity associated with executive functions. Finally, our third aim will confirm in behaving animals preferential activatio of COM or other 5-HT-excited neuron populations by 2A agonists, fear conditioning, and extinction of conditioned fear. These aims are innovative in combining anatomical, physiological, optogenetic, and behavioral approaches to characterize selective modulation of cellular components and synaptic connections underlying functionally defined cortical output channels. The results will be significant in providing a framework for understanding the functional role of 5 HT in regulating the output of cortical circuits. The new knowledge gained will provide insight into how 5-HT facilitates normal cognition and behavior, and why dysregulation of serotonergic signaling in the cerebral cortex leads to the behavioral deficits observed in schizophrenia, depression, and other mental health disorders.